Target nucleic acid and capture method

ABSTRACT

The present invention provides a rapid and sensitive method for assaying nucleic acids by means of hybridization techniques, wherein the detector probes are modified primers being incorporated into copies of the target nucleic acid before the hybridization reaction and a reagent combination as well as a kit therefor. The invention also provides a method for assaying nucleic acids by means of hybridization techniques, wherein the capturing probes are modified primers being incorporated into copies of the target nucleic acids before the hybridization reaction.

This application is a continuation of application Ser. No. 08/500,695 ,filed on Jul. 11, 1995, now abandoned, which is a division ofapplication Ser. No. 07/610,470 , now U.S. Pat. No. 5,476,769, filed onNov. 7, 1990, which is a continuation of application Ser. No.07/024,604, filed on Mar. 11, 1987, abandoned.

TECHNICAL FIELD

The present invention relates to a rapid and sensitive method forassaying nucleic acids by means of hybridization techniques, wherein thedetector probes are modified primers being incorporated into copies ofthe target nucleic acid before the hybridization reaction and a reagentcombination as well as a kit therefore.

Moreover, the invention relates to a method for assaying nucleic acidsby means of hybridization techniques, wherein the capturing probes aremodified primers being incorporated into copies of the target nucleicacids before the hybridization reaction and a reagent combination aswell as a kit therefore.

BACKGROUND OF INVENTION

In hybridization reactions a labelled oligo- or polynucleotide, i.e. theprobe is allowed to base-pair with the nucleic acid target. Varioushybridization methods have been used for the detection of nucleic acids.In direct hybridization methods the specimen is either in solution orfixed to a solid carrier. The nucleic acid which is to be identified isdemonstrated using one labelled probe.

In U.S. Pat. No. 4,486,539 a sandwich hybridization method has beendescribed. In this method two separate probes are used, one being adetector probe labelled and used for detection and the other being acapturing probe immobilized to a solid carrier for the separation of thetarget nucleic acid from the reaction mixture.

The method of hybridization in solution is described in British PatentPublication No. 2 169 403. Two different probes both being in the samesolution phase are used in this method. The detector probe is labelledwith a detectable label and to the capturing probe a moiety havingaffinity for another component is attached. After the hybridization thehybrid formed between the capturing probe, target nucleic acid and thedetector probe, may be separated from the hybridization solution by theaid of the other moiety of the affinity pair.

The enzyme catalyzed polymerization of DNA where the nucleotide sequenceof a previously existing nucleic acid strand, i.e. the template isaccurately copied into its complementary strand, is well-known in theart and has been described e.g. in Kornberg, DNA replication, W. H.Freeman & Co, San Francisco, pp. 221-225 and 670-679, 1980 and Maniatiset al., Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, p. 122, 1982. This biological multiplication is used inhybridization assays in which the microbe to be detected is cultivatedand hence its DNA enriched prior to the test and is described e.g. inWoo, Methods Enzymol. 68, p. 389, 1979 and in U.S. Pat. No. 4,358,535.Specific DNA sequences can also be amplified within living cells e.g. bythe use of suitable drugs as described by Clewell and Helinski in J.Bacteriol. 110, p. 1135, 1972 and in European Patent Application No. 55742. A more specific DNA-enrichment is described in in the EuropeanPatent Application No. 175 689 in which the target is linked to aplasmid replicon and introduced into a suitable cell. Yet another methodis described in the European Patent Application No. 201 184, in whichthe primer dependence of DNA synthesis is utilized to create an in vitroreaction for the amplification of the target DNA. In the European PatentApplication No. 200 362 a method for detecting amplified genes issuggested.

SUMMARY OF INVENTION

In the hybridization method of the present invention either the detectorprobes or the capturing probes act as modified primers beingincorporated into the copies of the target nucleic acid in a templatedependent polymerization process before the hybridization reaction.

In the method of the invention at least one primer is needed and theprimers are always modified. If the detector probes act as primers inthe polymerization reaction, the primers are provided with at least onesuitable, detectable label or at least one specific site whereto atleast one suitable, detectable label can be attached.

Alternatively the capturing probes can be used as primers in thepolymerization reaction, in which case the primers are provided with atleast one suitable moiety of an affinity pair or at least one sitewhereto at least one suitable moiety of an affinity pair can beattached.

The invention also discloses a reagent combination and a kit comprisingin packaged form a multicontainer unit comprising the reagentcombination needed for the performance of the test.

By using the detector or capturing probes as primers in a polymerizationreaction it is possible to increase the sensitivity of the hybridizationreaction by several orders of magnitude compared with methods measuringthe target directly. Furthermore, the invention provides a convenientmethod to perform the hybridization reaction in solution so that thehybrids are easily and rapidly separated from the hybridization solutionafter the hybridization reaction.

The method of the invention is convenient for diagnosting certaindiseases, which are very difficult to diagnose with conventionalmethods. Thus the method is especially useful for the identification ofcytomegalovirus and the HI- or AIDS-virus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the base sequence of the modified primers, P_(a) andP_(b), as well the selective probes, S₁ and S₂, used in Example 1, 2 and3, as well as the relative sites of the modified primers, P_(a) andP_(b), and the selective probes, S₁ and S₂, on the target nucleic acidin question. The long lines, A and B, indicate the two target strandswhich continue in both directions. The arrowheads on the primers, P_(a)and P_(b), indicate the direction in which they are elongated in thepolymerization process.

FIG. 2 represents the base sequence of the modified primers, P_(a) andP_(b), and the selective probe, S used in Example 4, as well as therelative sites of the modified primers, P_(a) and P_(b) and theselective probe S on the target nucleic acid in question. Line Aindicates the RNA and its identical DNA copies and line B shows thecomplementary DNA copies. The arrowheads on the primers P_(a) and P_(b)indicate the direction in which they are elongated in the polymerizationprocess.

DETAILED DESCRIPTION OF THE INVENTION

Preparation of assay material.

The probes used in the method are oligo- or polynucleotides. The probescan be prepared synthetically or semi-synthetically, which are thepreferred mode to prepare probes, which will act as primers, too. It isalso quite possible to prepare the probes by recombinant techniques, orfrom nucleic acids isolated directly from nature. A probe may be boundto a suitable vector. It may contain vector parts or be completelydevoid of vector parts. Actually a multitude of suitable primers andprobes, which can be used, are commercially available.

The detector probes as modified primers.

In one of the methods of the present invention the detector probes areoligonucleotides or polynucleotides, which can be bound to the targetnucleic acid by base-pairing and which can act as primers for a templatedependent nucleic acid synthesizing enzyme. It is essential that thedetector primers are provided with at lest one suitable, detectablelabel or at least one specific site whereto at least one suitabledetectable label can be attached.

Various radioactive isotopes or radioactively labelled compounds may beused as labels. The label substance may also be fluorescent,luminescent, light emitting, enzymatically or immunologicallydemonstrable etc. Labels based on the affinity of biotin and avidin orstreptavidin, lanthanide chelates, ferritin and heme compounds, andimmunologically demonstrable haptens such as AAF and AIF(acetoxyacetylfluorene derivatives) can be mentioned as examples.Identification by the aid of mediators, for example proteins, is alsopossible.

The method according to the invention is not dependent on the labelused. All currently known label substances suitable for nucleic acidhybridization can be freely applied to the method. It is, however,essential that if the detector probes act as primers, the label isselected from a group of labels, which will not disturb the function ofthe primer. The label has to be attached to the detector primer in sucha way that the nucleic acid polymerizing enzyme still can recognize itas a primer.

The capturing probes as primers.

In the other method of the present invention the capturing probes areoligonucleotides or polynucleotides which can be bound to the targetnucleic acid by base-pairing and which can act as primers for a templatedependent nucleic acid synthesizing enzyme. It is essential that thecapturing primers are provided with at least one suitable moiety of anaffinity pair or at least one site whereto at least one suitable moietyof an affinity pair can be attached. It is also possible to attach themoiety or moieties of the affinity pair through a mediator to thecapturing primer. The only conditions are that it is possible toseparate the hybrid from the hybridization solution by the aid of theaffinity pair and that the primer function is not harmed.

It is not necessary to attach the moiety or moieties of the affinitypair to the capturing primer at the beginning of the polymerization. Itmay be added at any time after the polymerization process to themodified primer having been incorporated into the copies of the targetnucleic acid.

The moiety of the affinity pair is a component having affinity foranother component. For example, biotin--avidin or streptavidin, a heavymetal derivative--a thiogroup, various homopolynucleotides such as polydG --poly dC, poly dA--poly dT, and poly dA--poly U, are such affinitypairs. But also other component pairs can be used, provided that theyhave affinity strong enough to allow their specific binding to theaffinity column. Suitable affinity pairs are found among ligands andconjugates used in immunological methods.

The selective capturing probe.

In one of the methods of the present invention, wherein the detectorprobes act as primers, a selective capturing probe is required to allowthe selective separation of the modified primers being incorporated intothe copies of the target nucleic acids. It is essential that thecapturing probes are sufficiently homologous to the target nucleic acidto allow specific hybridization and thereby selective separation of thedetector primers having been incorporated into the copies of the targetnucleic acid.

The selective detector probe.

In the other method of the present invention, wherein the capturingprobes act as modified primers, a selective detector probe is requiredto allow the detection of the modified primers being incorporated intothe copies of the target nucleic acids. It is essential that thedetector probe is sufficiently homologous to the target nucleic acid tohybridize specifically and thereby to identify the target nucleic acidselectively. The detector probes can be provided with any suitable,detectable labels for example with those mentioned above.

Reagent combinations.

The detector probe as a modified primer.

The present invention relates to a reagent combination comprising atleast one modified primer, provided with at least one suitable,detectable label or at least one specific site whereto at least onesuitable, detectable label can be attached and at least one selectivecapturing probe provided with at least one moiety of an affinity pair orat least one specific site whereto at least one moiety of an affinitypair can be attached.

The capturing probe as a modified primer.

The present invention relates also to a reagent combination comprisingat least one modified primer provided with at least one suitable moietyof an affinity pair or at least one specific site whereto at least onesuitable moiety of an affinity pair can be attached and at least oneselective detector probe provided with at least one suitable, detectablelabel or at least one specific site whereto at least one suitable,detectable label can be attached.

Kits.

The present invention also discloses a convenient kit for assayingnucleic acids. The kit comprises in packaged form a multicontainer unitin which one of the reagent combinations mentioned above is combinedwith at least one of the following reactants or facilities needed in thetest i.e. optionally a container comprising at least one templatedependent polymerization agent, optionally a container with the fourdeoxynucleoside triphosphates, optionally a suitable facility for thepolymerization and the hybridization process, optionally a suitablefacility for the separation of the copies of the target nucleic acidsand optionally a suitable facility for assaying the label. The preferredfacilities and reactants are described in more detail in the followingpart of the specification.

The method of the invention

The preferred method of the present invention starts by adding at leasttwo modified primers, both primers being either detector or capturingprimers, to a denaturated sample solution. The modified primers willeach anneal to their complementary strand of the target nucleic acid,i.e. the template and upon addition of a template dependent nucleic acidsynthesizing enzyme the primers will be elongated. The process proceedsefficiently in vitro creating new nucleic acid strands which may beseveral thousand bases in length, provided the conditions are suitable.

By using an excess of modified primers the process may be repeated tocreate complementary copies to the newly synthesized strands, which thusare identical copies of the first template. By repeating this process acascade reaction is initiated whereby the target nucleic acid ismultiplied. The process may be repeated as many times as desired, toobtain the desired detection sensitivity. In cases where theconcentration of target nucleic acid is not extremely low onemultiplication is sufficient to make the target nucleic acid detectable.

It is also possible to use only one modified primer in the method of theinvention. In this case the multiplication is, however, not so efficientas by using at least two primers because the reaction is not a cascadetype reaction.

Both DNA and RNA can be determined by the method of the presentinvention. However, if the target nucleic acid is RNA it is mostconvenient first to copy the RNA to the corresponding cDNA by reversetranscriptase enzyme, whereafter the process continues as describedabove.

After the modified primers are incorporated into the copies of thetarget nucleic acids, a suitable selective probe recognizing the targetsequence and its copies is added to the reaction mixture and thehybridization reaction is performed under conditions suitable for therespective hybridization process chosen.

In the hybridization reaction, depending on the choice of modifiedprimers, either a selective capturing probe or a selective detectorprobe is allowed to hybridize with the copies of the target nucleic acidnow present in multifolded amounts compared to the amount of the targetnucleic acid in the original situation.

If the original sample contained the target sequence the added selectiveprobe will hybridize to newly synthetized copies of the target nucleicacid. A hybrid is formed between the modified primer-target molecule andthe selective probe. The hybrids formed are according to the presentinvention, conveniently separated from the hybridization solution by theaid of the moiety of the affinity pair, which is attached, either on thecapturing primer or on the selective capturing probe. Duringfractionation these capturing moiety containing hybrids adhere to asolid carrier by the aid the other moiety of the affinity pair and theamount of selective detector probe or detector primer adhering to thecarrier can be measured by conventional methods directly from thecarrier or after elution from the eluate. The amount of label is ameasure of the amount of target nucleic acid.

Before the fractionation, the solution is diluted, when necessary, torender the conditions advantageous for the affinity pair. Thereafter thesolution is contacted with the solid carrier. The carrier in questionmay be for instance an affinity chromatography column, a filter, aplastic surface or a glass surface. Convenient facilities for performingthe separation are different types of microtiter plates, dipsticksystems or magnetic particles, but it is quite possible to perform theseparation in test tubes and on beads etc.

The carrier material of the affinity column may be for instance,cellulose, latex, polyacrylamide, polystyrene, dextran or agarose. Thesematerials can also be used as suspensions in a test tube. It is alsoadvantageous to use test tubes having the other moiety of an affinitypair fixed to its inner surface. It is a prerequisite for the materialselected that it is possible to fix to it a component having affinity tothe moiety of the affinity pair which is attached to the capturingprimer or the selective capturing probe.

The separation of hybrid by the aid of one moiety of an affinity pairmust not necessarily be made using an affinity pair coupled to a solidcarrier. Whenever the chemical or physical behaviour of the modifiedprimer-target-selective probe complex differs enough from that of thefree detector primer or from the free selective detector probe it can beseparated from them for example by using electric or magnetic fields,phase extraction or precipitation.

If the detector probes act as modified primers incorporated into thecopies of the target nucleic acid, the hybrid can be separated from thereaction mixture by the aid of selective capturing probes immobilized onsolid carriers. In this method the rate limiting step is created whenthe target nucleic acid and its copies, wherein detector primers havebeen incorporated, must hybridize with the selective capturing probethat is immobilized on a solid carrier. Therefore, the hybridization insolution is a more preferred method of the invention than this. However,if the method is carried out by using an immobilized capturing probe,the hybrid formed on the solid carrier is washed and the amount of thelabel on the carrier is measured by conventional methods.

EXAMPLE 1

Detection of cytomegalovirus DNA by using detector probes as modifiedprimers

The principle of the test is demonstrated in Example 1. In this modelexperiment the target is a recombinant plasmid (pBR322/CMV HindIII L)which harbours a 12.3 kb fragment of the cytomegalovirus (CMV, AD 169,ATCC VR-538) genome. The two detector primers (P_(a), P_(b), FIG. 1)used were 20 nucleotides long and synthesized with standard methods onan automated synthesizer. They corresponded to two regions on theCMV-specific insert which were 111 nucleotides apart. Two selectivecapturing probes (S₁, S₂, FIG. 1) recognized regions on each of the twostrands between the two detector primers. The detector primers Pa andP_(b) were labelled with ³² P at their 5' ends to a specific activity of4×10⁹ CPM/μg using the well known reaction with polynucleotide kinaseand gamma-³² P-ATP (Maniatis et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory, 1982).

Biotinvlated nucleotides were added to the 3' ends of the capturingprobes using bio 11-dUTP and terminal transferase as described by Rileyet al., DNA, 5 (4), pp. 333-337, 1986. The target plasmid was linearizedby cleavage with the restriction enzyme EcoRI. DNA polymerase, Klenowfragment was purchased from Boenringer-Mannheim and streptavidin agarosefrom.

Using these reagents the following experiment was performed.

Four different reactions were assembled containing 0, 10⁴, 10⁶ and 10⁸molecules (corresponding to 0, 2×10⁻²⁰, 2×10⁻¹⁸ and 2×10⁻¹⁶ moles)respectively of the target plasmid. In addition all four reactionscontained in a total volume of 50 μl: 2 pmol each of the two primers, 0,5 mM of each of the four deoxynucleoside triphosphates (i.e. dATP, dCTP,dGTP and dTTP), 10 mm Tris-Cl pH 7, 5 10 mM MgCl₂, 50 mM NaCl and 10 mMdithiothreitol. The mixture was heated to 100° C. for 2 min, thenincubated for 5 min at 37° C., whereafter 1 μl (equalling 1 unit) ofDNA-polymerase was added. Then the mixture was again incubated for 10min at 37° C. The boiling followed by annealing of the detector primersand an incubation with added DNA polymerase at 37° C. constitutes a DNAsynthesizing cycle.

In this experiment the cycle was either performed only once or repeated5 or 15 times. After the last cycle the sample was again heated to 100°C. whereafter 10 pmole of the selective capturing probe was addedtogether with NaCl, (0, 9 M) EDTA (20 mM) sodium phosphate pH 7, 5 (20mM) and sodium dodecyl sulphate (0.1%). The volume increased to 100 μland the concentrations given are as final concentrations. The mixturewas then incubated at 50° C. for 1 h. After this hybridization reaction200 μl of a 25% suspension of streptavidin-agarose in 1 M NaCl, 20 mMsodium phosphate, pH 7, 5, 1 mM EDTA was added. Biotinylated moleculeswere allowed to bind to the streptavidin-agarose for 15 min at 37° C. ina rotating mixer. The agarose was collected by brief centrifugation andthe supernatant removed by aspiration. The agarose was then washed oncein the buffered 1 M NaCl and twice in a solution containing 150 mM NaCl,15 mM sodium citrate 0.2% sodium dodecyl sulphate (pH 8) at 37° C. Theradioactivity of the agarose to which the formed hybrids were bound wasthen determined in a radioactivity counter. The harvesting and washingprocedure for DNA hybrids containing a biotinylated marker arepreviously known procedures described e.g. in British Patent PublicationNo. 2 169 403.

The results of the experiment are shown in Table 1. It is seen that onecycle of DNA synthesis incorporates enough radioactivity for detectiononly if high target concentrations are present, but that even the verylow target amount is detectable after 15 cycles. With high amount oftarget and 15 cycles the amount of detector primer became limiting.

                  TABLE 1                                                         ______________________________________                                                   32 P-activity in collected hybrids .sup.a)                         Amount of  (CPM above background) .sup.b)                                     target (moles)                                                                           1      5         15    No. of cycles                               ______________________________________                                        0           0       0         0                                               2 × 10.sup.-20                                                                     ND     ND         650                                              2 × 10.sup.-18                                                                     ND      300      11000                                             2 × 10.sup.-16                                                                     700    13000     36000                                             ______________________________________                                         .sup.a) Mean of two determinations                                            .sup.b) ND  not detectable (less radioactivity than 2 times mean              background activity)                                                     

EXAMPLE 2

Determination of cytomegalovirus DNA by using capturing probes asmodified primers.

In this example the capturing probes act as primers. The reagents usedwere the same as in Example 1 with the following exceptions: Thecapturing primers (P_(a), P_(b), FIG. 1) were not labelled with ³² P buttheir 5' ends were instead modified to contain a biotin residue. Thischemical modification was done using known methods described by Cholletand Kawashima, Nucleic Acids Research, 13, pp. 1529-1541, 1985. The twoselective probes (S₁ and S₂, FIG. 1) were in this case labelled in their5' ends to act as detector probes. Their specific activities wereapproximately 2×10⁹ and 2, 5×10⁹ cpm/μg respectively.

The reaction mixtures were assembled as described in Example 1. Thebiotinylated capturing primers were, however, added in 10 fold amounts,i.e. 20 pmol each per reaction. 1, 5 or 15 cycles were performed asdescribed whereafter the samples were heated to 100° C. and 0, 5 pmoleach of the ³² P-labelled probes S₁ and S₂ were added. The hybridizationwas carried out in the same conditions as described in Example 1.

The hybrids were then collected on streptavidin-agarose, washed andcounted for ³² P-activity, as in Example 1. The result is shown in Table2.

                  TABLE 2                                                         ______________________________________                                                   .sup.32 P-acitivity in collected hybrids .sup.a                    Amount of  (CPM above background).sup.b)                                      target (moles)                                                                           1      5         15    No. of cycles                               ______________________________________                                        0           0       0         0                                               2 × 10.sup.-20                                                                     ND     ND         800                                              2 × 10.sup.-18                                                                     ND      400      13000                                             2 × 10.sup.-16                                                                     300    11000     54000                                             ______________________________________                                         .sup.a) Mean of two determinations                                            .sup.b) ND  Not detectable (cf ex 1)                                     

EXAMPLE 3

Detection of cytomegalovirus DNA from clinical samples by usingcapturing probes as modified primers.

In this example the applicability of the method for the study ofclinical samples is demonstrated by detecting CMV from the urine of aninfant known to suffer from cytomegalovirus infection. The urine from ahealthy child was included as a control. Both samples were 10 ml ofurine from which the total DNA was isolated as described in Virtanen etal., J. Clin. Microbiol., 20 (6), pp. 1083-1088, 1984. The DNAS,dissolved into 20 μl H₂ O were used as target in reactions whichotherwise were performed as described in Example 2. After 10 cycles ofDNA-synthesis the labelled selective probe was added to the sample,allowed to hybridize, and the hybrids collected. The DNA from the urineof the patient showed a clearly elevated radioactivity in hybrids whilethat from the healthy person showed background radioactivity only. Theactual cpm-values were 2300 and 240 respectively.

EXAMPLE 4

Detection of Semiliki Forest virus RNA by using capturing probes asmodified primers.

Example 4 is to demonstrate that the method described also can be usedfor the detection of RNA. The model used was the RNA from Semliki Forestvirus (SFV).

The reagents used were two 5' biotinylated capturing primers (FIG. 2)(prepared as described in Example 2), a single 5' ³² P-labelledselective detector probe (prepared as described in Example 1), reversetranscriptase (Promega Biotech, and DNA polymerase, Klenow fragment(Boehringer Mannheim).

The first step in the detection of the SFV-RNA was to synthesize a cDNAcopy. The 20 μl reaction mixture contained 10 mM tris-Cl pH 8.3, 50 mMKCl, 10 mM MgCl₂, 10 mM dithiothreitol 0, 5 mm each of the fourdeoxynucleoside triphosphates, 0, 5 μg t-RNA, 10 pg of SFV-RNA, 10 pmolof capturing primer P_(a) and 100 U reverse transcriptase. This mixturewas incubated at 37° C. for 15 min. Then the mixture was heated to 100°C. for 5 min and cooled to ambient temperature. Thereafter 50 μl of asolution containing 10 mM Tris-Cl pH 7, 4, 50 mM NaCl, 10 mM MgCl₂, 10mM dithiothreitol, 10 pmol of the capturing primer P_(b) and 0, 5 mM ofeach of the 4 deoxynucleosidetriphosphates. The temperature was elevatedto 37° C. and after 5 min 1 U of DNA-polymerase was added. After anadditional 10 min incubation the reaction mixture was incubated at 100°C. for 5 min, the mixture was cooled to 37° C. and 5 cycles of DNAsynthesis was performed. After a final denaturation step 0.1 pmol (1,2×10⁶ cpm) of the selective detector probe was added in 80 μl M NaCl, 50mM EDTA, 50 mM sodiumphospate (pH 7, 5) 0.1% sodium dodecyl sulphate.The solution was then incubated for 2 h at 55° C. whereafter the hybridswere collected and washed as described in Example 1.

As a negative control for the reactions an identical sample was giventhe same treatment except for that no reverse transriptase was added.The sample in which the RNA was converted to cDNA with reversetranscriptase yielded 420 cpm ³² P-activity in captured hybrids, whilethe negative control yielded 50 cpm.

What is claimed is:
 1. A method for assaying for a target nucleic acidcomprising the steps of(a) providing to a sample which comprises atarget nucleic acid a capturing primer, in which the capturing primercomprises at least one moiety of an affinity pair or at least onespecific site to which at least one moiety of an affinity pair can beattached; (b) exposing said capturing primer to the target nucleic acid,in single-stranded form, under conditions wherein a template dependentpolymerization reaction occurs, and a complementary copy of thesingle-stranded target nucleic acid is formed which comprises thecapturing primer; (c) exposing the complementary copy of thesingle-stranded target nucleic acid to a detector probe having asequence which selectively hybridizes to the complementary copy of thesingle-stranded target nucleic acid, under conditions wherein ahybridization reaction occurs; (d) binding the capturing primercomprised in the complementary copy of the single-stranded targetnucleic acid to a retrievable form of the other moiety of the affinitypair; (e) separating the complementary copy of the single-strandedtarget nucleic acid hybridized to the detector probe from the remainderof the sample by retrieving the bound affinity pair; and (f) detectingthe presence of the hybridized nucleic acids with the aid of thedetector probe.
 2. The method as claimed in claim 1, wherein the targetnucleic acid comprises a first strand and a second strand, and twocapturing primers are provided according to step (a), in which one ofsaid capturing primers hybridizes to the first strand of the targetnucleic acid and the other capturing primer hybridizes to the secondstrand of the target nucleic acid.
 3. The method as claimed in claim 1,wherein at least one moiety of an affinity pair is attached to thecapturing primer after the polymerization reaction of step (b) butbefore binding the capturing primer of step (d).
 4. A method accordingto claim 1, wherein the target nucleic acid is cytomegalovirus DNA.
 5. Amethod according to claim 1, wherein the target nucleic acid is Semlikiforest virus DNA.
 6. A method for assaying for a double-stranded targetnucleic acid comprising the steps of(a) providing to a sample comprisinga double-stranded target nucleic acid a plurality of capturing primers,in which the capturing primers each comprise at least one moiety of anaffinity pair or at least one specific site to which at least one moietyof an affinity pair can be attached; (b) denaturing the double-strandedtarget nucleic acid; (c) exposing said capturing primers to the targetnucleic acid, in single-stranded form, under conditions wherein atemplate dependent polymerization reaction occurs, whereby complementarycopies of the single-stranded target nucleic acid are formed whichcomprise the capturing primers; (d) exposing the complementary copies ofthe single-stranded target nucleic acid to a plurality of detectorprobes, each detector probe having a sequence which selectivelyhybridizes to a complementary copy of the single-stranded target nucleicacid, under conditions wherein a hybridization reaction occurs; (e)binding the capturing primers comprised in the complementary copies ofthe single-stranded target nucleic acid to a plurality of the othermoiety of the affinity pair in retrievable form; (f) separating thecomplementary copies of the single-stranded target nucleic acidhybridized to the detector probes from the remainder of the sample byretrieving the bound affinity pairs; and (g) detecting the presence ofthe hybridized nucleic acids with the aid of the detector probes.
 7. Amethod for assaying for a double-stranded target nucleic acid comprisingthe steps of(a) providing to a sample comprising a double-strandedtarget nucleic acid a plurality of capturing primers, in which thecapturing primers each comprise at least one moiety of an affinity pairor at least one specific site to which at least one moiety of anaffinity air can be attached; (b) denaturing the double-stranded targetnucleic acid; (c) exposing said capturing primers to the target nucleicacid, in single-stranded form, under conditions wherein a templatedependent polymerization reaction occurs, whereby a complementary copyof the single-stranded target nucleic acid is formed which comprises acapturing primer; (d) denaturing the complementary copy from thesingle-stranded target nucleic acid; (e) repeating steps (b) and (c)until a plurality of complementary copies of the single-stranded targetnucleic acid have been produced; (f) exposing the complementary copiesof the single-stranded target nucleic acid to a plurality of detectorprobes, each detector probe having a sequence which selectivelyhybridizing to a complementary copy of the single-stranded targetnucleic acid, under conditions wherein a hybridization reaction occurs;(g) binding the capturing primers comprised in the complementary copiesof the single-stranded target nucleic acid to a plurality of the othermoiety of the affinity pair in retrievable form; (h) separating thecomplementary copies of the single-stranded target nucleic acidhybridized to the detector probes from the remainder of the sample byretrieving the bound affinity pairs; and (i) detecting the presence ofthe hybridized nucleic acids with the aid of the detector probes.
 8. Themethod as claimed in claim 6, in which some capturing primers bind toone strand of the target nucleic acid and other capturing primers bindto the other strand of the target nucleic acid.
 9. The method as claimedin claim 7, in which some capturing primers bind to one strand of thetarget nucleic acid and other capturing primers bind to the other strandof the target nucleic acid.